Corneodesmosin based test and model for inflammatory disease

ABSTRACT

The present invention relates to a polynucleotide encoding the corneodesmosin protein having one or more nucleotide insertions, deletions, or substitutions at one or more novel positions. The invention also relates to the corneodesmosin protein having one or more amino acid insertions, deletions, and substitutions. These nucleotide and amino acid polymorphisms are useful in diagnosing or determining susceptibility to corneodesmosin-mediated disease, such as inflammatory diseases, including psoriasis, and in treating such disease. Host cells and transgenic non-human animals comprising polynucleotides or proteins of the invention are provided. Methods of screening for agents for use in treating corneodesmosin-mediated disease are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 10/204,884, filed Jan.29, 2003, which is the National Stage of International Application No.PCT/GB01/00795, filed Feb. 23, 2001, which claims priority from GB0004312.5, filed Feb. 23, 2000.

FIELD OF THE INVENTION

The present invention relates to nucleotide substitutions, deletions, orinsertions in the corneodesmosin gene, and the exploitation of thesepolymorphisms in the detection and/or treatment ofcorneodesmosin-mediated disease, such as inflammatory diseases,including psoriasis. The present invention also relates topolynucleotides encoding the corneodesmosin protein having one or morenucleotide polymorphisms, and to proteins encoded by saidpolynucleotides. Also provided are transgenic non-human animalscomprising the polynucleotides of the present invention, and methods andkits for treating, diagnosing, or determining susceptibility tocorneodesmosin-mediated disease, in particular by way of gene therapy.

BACKGROUND OF THE INVENTION

In recent years, it has been recognized that there is considerablegenetic diversity in human populations, with common polymorphismsoccurring on average at least every kilobase in the genome.Polymorphisms which affect gene expression or activity of the encodedgene product may account for susceptibility to, or expression of,disease conditions, either directly or through interaction with othergenetic and environmental factors.

Understanding the molecular basis for disease, by sequencing the humangenome and characterizing polymorphisms, will enable the identificationof those individuals at greatest risk of disease. This will allow thebetter matching of treatment and disease, and enable the production ofnew and improved targets for drugs. Screening and treatment of diseasemay also be better targeted to those in need, thus increasing thecost-effectiveness of health-care provision.

One area in need of such approaches is the diagnosis and treatment ofinflammatory diseases. Inflammation, which can be broadly defined as thedestructive sequelae to activation of elements of the body's immunesystem, is a feature of many diseases including infection, autoimmunedisorders, and benign and malignant hyperplasia. The identification ofgenetic factors which influence susceptibility to such disorders willprovide important new insights into inflammatory disease, and may yieldimportant new diagnostic and/or prognostic tests and treatments.

Psoriasis is a chronic inflammatory cutaneous disorder which affectsapproximately 2% of the population in the UK and US. Psoriasis manifestsitself as red scaly skin patches, principally on the scalp, elbows andknees, and is caused by epidermal hyperproliferation, and abnormaldifferentiation and infiltration of inflammatory cells. Psoriasis mayalso be associated with other inflammatory diseases such as arthritis,Crohn's disease, and HIV infection. Population, family, and twin studiesall suggest an important genetic component in the pathogenesis ofpsoriasis, coupled with environmental triggers such as streptococcalinfection and stress.

Psoriasis is one of a number of autoimmune diseases that displaysignificant human leukocyte antigen (HLA) associations. The analysis ofpopulation-specific HLA haplotypes has provided evidence thatsusceptibility to psoriasis is linked to the class I and II majorhistocompatibility complexes (MHC) on human chromosome 6 (Jenisch et al.(1998) Am. J. Hum. Genet 63:191-199). These studies show that psoriasisconsists of two distinct disease subtypes (Type I and Type II), whichdiffer in age of onset and in the frequency of HLA types. Type Ipsoriasis has an age of onset of prior to 40 years and HLA types Cw6,B57, and DR7 are strongly increased. Patients with Type I psoriasis aremuch more likely to have a positive family history for the disease. Incontrast, only about 10% of Cw6-positive individuals develop Type IIpsoriasis disease, with HLA-Cw2 being over-represented in this group.

Linkage analysis and association studies suggest the presence of a majorgenetic determinant of psoriasis within the MHC, the strongest candidategene marker being HLA-C. The most significant association has been shownbetween HLA-Cw6 and disease Type IA, which has the earliest onset ofdisease at 0 to 20 years. However, specific involvement of the HLA-Cw6genotype in disease pathogenesis has yet to be established.

Recently, attention has focused on non-HLA genes close to HLA-C, inparticular the corneodesmosin gene (also known as the S gene), which islocated approximately 160 kb telomeric of the HLA-C locus. Thecorneodesmosin gene consists of 2 exons spanning approximately 5.3 kb ofgenomic DNA sequence. Two corneodesmosin mRNAs of 2.2 kb and 2.6 kb,resulting from alternative splicing, have been described (Guerrin et al.(1998) J. Biol. Chem. 273:22640-22647). Association studies (Ahnini etal. (1999) Hum. Mol. Genet. 8:1135-1140) suggest a strong, significantassociation between a polymorphism at position 1243 of thecorneodesmosin gene and psoriasis. A corneodesmosin gene haplotype wassubsequently defined, which by TDT analysis was shown to have a strong,significant association with psoriasis (Allen et al. (1999) Lancet353:1589-90).

In human epidermis and other cornified squamous epithelia,corneodesmosin is located in the desmosomes of the upper living layers,and in related structures of the cornified layers, the corneodesmosomes.During maturation of the cornified layers, the protein undergoes aseries of cleavages, thought to be a prerequisite of desquamation(shedding of the cuticle or epidermis). Corneodesmosin is detected as aglycosylated and phosphorylated basic protein with an apparent molecularmass of 52-56 kDa. During stratum corneum maturation, corneodesmosin isprogressively proteolysed until desquamation occurs. In superficialcorneocytes, the 52-56 kDa form is no longer detected and immunoreactivefragments of 45 to 30 kDa predominate. Since location, biochemicalcharacteristics and processing of corneodesmosin are similar in severalmammals, it is likely that the protein is essential for the function ofcorneodesmosomes and corneocyte cohesion. It has been shown thatexpression of the 56 kDa epidermal keratin polypeptide is increased inpsoriatic lesions compared with normal skin and transformation ofdesmosomes into corneodesmosomes is altered in psoriatic epidermis.

Psoriasis affects approximately 6.4 million people in the US and causesvarying ranges of physical discomfort, pain and disability. At present,the causes of psoriasis are unknown. There is no specific test forpsoriasis or susceptibility thereto, and diagnosis is based solely onclinical examination and skin histopathology.

It is likely that corneodesmosin is implicated in a range of skindiseases, including psoriasis. In this text, diseases in whichcorneodesmosin is implicated in the pathology will be referred to as“corneodesmosin-mediated disease.”

The present invention aims to overcome or ameliorate previouslimitations in the art by providing means and methods for the detectionand treatment of individuals having, or being susceptible to,corneodesmosin-mediated disease, in particular inflammatory conditionssuch as psoriasis.

In a first aspect, the present invention provides an isolated orrecombinant polynucleotide comprising a nucleic acid sequence encodingthe corneodesmosin gene of FIG. 1 (SEQ ID NO:1), wherein said nucleicacid sequence comprises a nucleotide substitution, deletion or insertionat one or more of positions 6984, 7068, 7077, 7107, 7164, 8884, 8906,8931, 9538, 9607, 9608, 9647, 9667, 9745, 9761, 9926, 9952, 9968, 10082,10161, 10162, 10363, 11567, 11641, 11649, 11808, 11839, 11885, 11977,12018, 12136, 12149; 12198, 12283, 12318, 12345, 12373, 12901, 13001,13020, 13108, 13117, 13178, 13224, 13316, 13365, 13562, 13605, 13670,13859, 13889 and 13914 of FIG. 1 (corresponding to positions 284, 368,377, 407, 464, 2184, 2206, 2231, 2838, 2907, 2908, 2947, 2967, 3045,3061, 3226, 3252, 3268, 3382, 3461, 3462, 3663, 4867, 4941, 4949, 5108,5139, 5185, 5277, 5318, 5436, 5449, 5498, 5583, 5618, 5645, 5673, 6201,6301, 6320, 6408, 6417, 6478, 6524, 6616, 6665, 6862, 6905, 6970, 7159,7189 and 7214 of SEQ ID NO:1). These novel polymorphisms in thecorneodesmosin gene, at the positions indicated above, may beresponsible for corneodesmosin-mediated disease. In particular, thepolymorphisms of the present invention may be useful in identifyingindividuals susceptible or resistant to corneodesmosin-mediated disease,and in the diagnosis or treatment of such conditions. Preferredcombinations of the polymorphisms of the invention are the haplotypesshown in Tables 10a and b. The most preferred haplotype is B of Table10a.

The polynucleotide of this invention is preferably DNA, or may be RNA orother options.

By “isolated” is meant a polynucleotide sequence which has been purifiedto a level sufficient to allow allelic discrimination. For example, anisolated sequence will be substantially free of any other DNA or proteinproduct. Such isolated sequences may be obtained by PCR amplification,cloning techniques, or synthesis on a synthesizer. By recombinant ismeant polynucleotides which have been recombined by the hand of man.

The corneodesmosin gene sequence shown in FIG. 1 (SEQ ID NO:1) refers tothe genomic clone of corneodesmosin, detailed in GenBank Accession No.AC006163 (a genomic clone of the MHC region on chromosome 6p21.3). Thesingle nucleotide polymorphisms of the invention are shown in bold typeand underlined on this figure, and have each been given a positionalreference with respect to this figure. For reference and comparison withprior art publications, the positional references with respect to thecoding sequence have also been given in Table 6, Column 2, wherenucleotide position 1 corresponds to the first nucleotide of exon 1 andnucleotides upstream of this are given a negative prefix.

A polymorphism is typically defined as two or more alternativesequences, or alleles, of a gene in a population. A polymorphic site isthe location in the gene at which divergence in sequence occurs.Examples of the ways in which polymorphisms are manifested includerestriction fragment length polymorphisms, variable number of tandemrepeats, hypervariable regions, minisatellites, di- or multi-nucleotiderepeats, insertion elements and nucleotide deletions, additions orsubstitutions. The first identified allele is usually referred to as thereference allele, or the wild type. Additional alleles are usuallydesignated alternative or variant alleles. Herein, the sequence exactlyas shown in FIG. 1 is designated the reference sequence, and is not partof the invention. Nucleic acid sequences of the present invention whichdiffer from the sequence of FIG. 1 (SEQ ID NO:1) at one or more of thepositions indicated above may be referred to as variants of FIG. 1 (SEQID NO:1).

A single nucleotide polymorphism is a variation in sequence betweenalleles at a site occupied by a single nucleotide residue. Singlenucleotide polymorphisms (SNP's) arise from the substitution, deletionor insertion of a nucleotide residue at a polymorphic site. Typically,this results in the site of the variant sequence being occupied by anybase other than the reference base. For example, where the referencesequence contains a “T” base at a polymorphic site, a variant maycontain a “C,” “G” or “A” at that site. Single nucleotide polymorphismsmay result in corresponding changes to the amino acid sequence. Forexample, substitution of a nucleotide residue may change the codon,resulting in an amino acid change. Similarly, the deletion or insertionof three consecutive bases in the nucleic acid sequence may result inthe insertion or deletion of an amino acid residue. For ease ofreference, where a single nucleotide polymorphism of the presentinvention results in the insertion or deletion of a nucleotide or aminoacid residue, the numbering system of FIGS. 1 (SEQ ID NO:1) and 2 (SEQID NO:2) have been maintained.

The single nucleotide polymorphisms of the present invention which occurwithin the protein coding sequence may contribute to the phenotype of anorganism by affecting protein structure or function. The effect may beneutral, beneficial or detrimental, depending upon the circumstances.Whatever the effect, the identification of such polymorphisms enablesfor the first time determination of susceptibility to disease, and newmethods of treatment. The single nucleotide, polymorphisms of theinvention which occur in the non-coding 5′ or 3′ untranslated regions,may not affect protein sequence, but may exert phenotypic effects byinfluencing replication, transcription and/or translation. Apolymorphism may affect more than one phenotypic trait or may be relatedto a specific phenotype. In the present invention, polymorphisms in thecorneodesmosin gene are likely to affect the phenotype of an individualwith respect to corneodesmosin-mediated disease, such as inflammatorydisease, in particular psoriasis.

The single nucleotide polymorphisms of the corneodesmosin gene,including those of the present invention, are listed in Table 6 where:

-   -   Column 1 designates each single nucleotide polymorphism a        reference number.    -   Column 2 provides the positional reference of the polymorphism        with respect to FIG. 1.    -   Column 3 indicates position of the SNP with respect to the        corneodesmosin coding sequence.    -   Column 4 shows the location of the polymorphisms in the gene.    -   Column 5 shows the sequence flanking the polymorphism, the        polymorphism itself being shown in bold type. For example, the        polymorphism at position 6984 is shown as C/T, meaning that the        variant sequence comprises a T residue, rather than the native C        residue.    -   Column 6 denotes the standard IUB code.    -   Column 7 denotes the SEQ ID NO of the corresponding flanking        sequence.

As discussed above, where a single nucleotide polymorphism of thepresent invention comprises a nucleotide substitution, the substitutionmay comprise the replacement of the reference base at a polymorphic sitewith any other base. Each single nucleotide polymorphism described inTable 6, Column 4, represents a preferred embodiment of the invention.

It will be appreciated by those skilled in the art that corneodesmosingene sequences of the invention may comprise one or more nucleotidesubstitutions, deletions or insertions in addition to one or more of thesingle nucleotide polymorphisms of the invention.

In a second aspect, fragments of the above polynucleotides are provided,which comprise one or more nucleotide substitutions, insertions ordeletions at one or more of the above mentioned positions of FIG. 1 (SEQID NO:1). Preferably, a fragment may comprise, or even consist of, thepolynucleotide sequence of Table 6, Column 4. The novelty of a fragmentaccording to the present embodiment. may be easily ascertained bycomparing the nucleotide sequence of a fragment with sequencescatalogued in databases such as GenBank, or by using computer programssuch as DNASIS (Hitachi Engineering, Inc.) or Word Search or FAST A ofthe Genetic Computer Group (Madison, Wis.).

Preferably, the fragments do not encode a full length protein, as isgenerally the case with the aforementioned polynucleotides, butotherwise satisfy the requirements of the first aspect. Preferredfragments may be 10 to 150 nucleotides in length. More preferably, thefragments are between 5 to 10, 5 to 20, 10 to 20, 20 to 50, or 50 to 100nucleotides in length. For example, the fragments may be 5, 8, 10, 12,15, 18, 20, 22, 25, 28, 30, or 35 nucleotides in length. The fragmentsmay be useful in a variety of diagnostic, prognostic or therapeuticmethods, or may be useful as research tools for example in drugscreening.

In a third aspect of the invention, there is provided non-coding,complementary sequences which hybridize to the corneodesmosin genesequence. Such “anti-sense” sequences are useful as probes or primersfor detecting an allele of a polymorphism of the invention, or in theregulation of the corneodesmosin gene. They may also be used as agentsfor use in the identification and/or treatment of individuals having orbeing susceptible to corneodesmosin-mediated disease.

The anti-sense sequences of the invention include those which hybridizeto an allele of a polymorphism of the invention, and also those whichhybridize a region flanking the polymorphic site to enable amplificationof an allele of one or more polymorphisms. These sequences may be usefulas probes or primers. To be useful as a probe, the antisense sequenceshould bind preferentially one allele of one or more polymorphisms ofthe present invention and will, preferably, comprise the exactcomplement of one allele of one or more polymorphisms of the invention.Thus, for example, where the variant comprises a “G” residue at position7068 of FIG. 1 (corresponding to position 368 of SEQ ID NO:1), it ispreferred that the anti-sense sequence will comprise a “C” residue. Suchanti-sense sequences which are capable of specific hybridization todetect a single base mismatch may be designed according to methods knownin the art and described in Maniatis et al. (1989) Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, NY, andBerger et al. (eds.) (1987) Methods in Enzymology, Vol. 152, Guide toMolecular Cloning Techniques, Academic Press Inc., San Diego, Calif.;Gibbs et al. (1989) Nuc. Acids Res. 17:2437; Kwok et al. (1990) Nuc.Acids Res. 18:999; and Miyada et al. (1987) Methods Enzymol. 154:94.Variation in the sequence of these anti-sense sequences is acceptablefor the purposes of the present invention, provided that the ability ofthe anti-sense sequence to distinguish between alleles of a polymorphismis not compromised. Similarly, variation in the sequence of a primersequence is acceptable, provided its ability to mediate amplification ofa polymorphic site is not compromised. Preferably, a primer sequencewill hybridize to the corneodesmosin gene under stringent conditionswhich are defined below.

In relation to the present invention, “stringent conditions” refers tothe washing conditions used in a hybridization protocol. In general, thewashing conditions should be a combination of temperature and saltconcentration so that the denaturation temperature is approximately 5 to20° C. below the calculated T_(m) of the nucleic acid under study. TheT_(m) of a nucleic acid probe of 20 bases or less is calculated understandard conditions (1M NaCl) as [4° C.×(G+C)+2° C.×(A+T)], according toWallace rules for short oligonucleotides. For longer DNA fragments, thenearest neighbor method, which combines solid thermodynamics andexperimental data may be used, according to the principles set out inBreslauer et al. (1986) PNAS 83:3746-3750. The optimum salt andtemperature conditions for hybridization may be readily determined inpreliminary experiments in which DNA samples immobilized on filters arehybridized to the probe of interest and then washed under conditions ofdifferent stringencies. While the conditions for PCR may differ from thestandard conditions, the T_(m) may be used as a guide for the expectedrelative stability of the primers. For short primers of approximately 14nucleotides, low annealing temperatures of around 44° C. to 50° C. areused. The temperature may be higher, depending upon the base compositionof the primer sequence used.

The anti-sense polynucleotides of this embodiment may be the full lengthof the corneodesmosin gene of FIG. 1 (SEQ ID NO:1), or more preferablymay be 5 to 200 nucleotides in length. Preferred polynucleotides are 5to 10, 10 to 20, 20 to 50, 50 to 100 or 100 to 200 nucleotides inlength. Primers, in particular, are typically 10 to 15 nucleotides long,and may occasionally be 16 to 25.

In a preferred embodiment, the polynucleotides of the aforementionedaspects of the invention may be in the form of a vector, to enable thein vitro or in vivo expression of the polynucleotide sequence. Thepolynucleotides may be operably linked to one or more regulatoryelements including a promoter; regions upstream or downstream of apromoter such as enhancers which regulate the activity of the promoter;an origin of replication; appropriate restriction sites to enablecloning of inserts adjacent to the polynucleotide sequence; markers, forexample antibiotic resistance genes; ribosome binding sites; RNA splicesites and transcription termination regions; polymerization sites; orany other element which may facilitate the cloning and/or expression ofthe polynucleotide sequence. Where two or more polynucleotides of theinvention are introduced into the same vector, each may be controlled byits own regulatory sequences, or all sequences may be controlled by thesame regulatory sequences. In the same manner, each sequence maycomprise a 3′ polyadenylation site. The vectors may be introduced intomicrobial, yeast or animal DNA, either chromosomal or mitochondrial, ormay exist independently as plasmids. Examples of suitable vectors willbe known to persons skilled in the art and include pBluescript II,LambdaZap, and pCMV-Script (Stratagene Cloning Systems, La Jolla (USA))Appropriate regulatory elements, in particular, promoters will usuallydepend upon the host cell into which the expression vector is to beinserted. Where microbial host cells are used, promoters such as thelactose promoter system, tryptophan (Trp) promoter system, β-lactamasepromoter system or phage lambda promoter system are suitable. Whereyeast cells are used, preferred promoters include alcohol dehydrogenaseI or glycolytic promoters. In mammalian host cells, preferred promotersare those derived from immunoglobulin genes, SV40, Adenovirus, BovinePapilloma virus, etc. Suitable promoters for use in various host cellswould be readily apparent to a person skilled in the art (see, forexample, Ausubel et al. (eds.), Current Protocols in Molecular Biology,published by Wiley).

In a fourth aspect of the present invention there is provided a proteinor protein fragment comprising an amino acid substitution, deletion orinsertion at one or more of positions 18, 130 or 180 of the amino acidsequence of FIG. 2 (SEQ ID NO:2). Preferably, the protein or proteinfragment is encoded by a polynucleotide according to the first aspect ofthe invention, and comprises a nucleotide insertion, deletion orsubstitution at one or more of positions 7164, 10082, 10161, 10162 and10363 of FIG. 1 (corresponding to positions 464, 3382, 3461, 3462 and3663 of SEQ ID NO:1, respectively). The corneodesmosin protein orprotein fragments of the invention may comprise one or morepolymorphisms in addition to one or more of the above-mentionedpolymorphisms of FIG. 2.

The amino acid sequence exactly as shown in FIG. 2 (SEQ ID NO:2) may bereferred to as the reference sequence, and is not part of the invention.The amino acid sequence of FIG. 2 (SEQ ID NO:2) having an amino acidsubstitution, deletion or insertion at one or more of the positionsindicated above may be referred to as a variant of FIG. 2 (SEQ ID NO:2).The reference amino acid at one or more of the above polymorphic sitesmay be replaced by any other amino acid residue to produce a variantsequence. Amino acid sequences of FIG. 2 (SEQ ID NO:2) having one ormore of the polymorphisms disclosed in Table 4 are each preferredembodiments of the invention.

Protein fragments may be functional or non-functional and may be usefulin drag screening or gene therapy. Functional fragments may be definedas those which have characteristics of the corneodesmosin protein. Thefragments may be at least 10, preferably at least 15, 20, 25 30, 35, 40or 50 amino acids in length.

In a fifth aspect of the present invention, there are providedantibodies which react with an antigen of a protein or protein fragmentof the fourth aspect. Antibodies can be made by the procedure set forthby standard procedures (Harlow and Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY). Briefly, purifiedantigen can be injected into an animal in an amount and in intervalssufficient to elicit an immune response. Antibodies can either bepurified directly, or spleen cells can be obtained from the animal. Thecells are then fused with an immortal cell line and screened forantibody secretion. The antibodies can be used to screen DNA clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced as described in, for example, Kelly et al. (1992)Bio/Technology 10:163-167 and Bebbington et al. (1992) Bio/Technology10:169-175. Preferably, the antigen being detected and/or used togenerate a particular antibody will include proteins or proteinfragments according to the fourth aspect.

In a sixth aspect of the present invention, there is provided host cellcomprising a polynucleotide according to any of the aforementionedaspects, for expression of the polynucleotide. The host cell maycomprise an expression vector, or naked DNA encoding saidpolynucleotides. A wide variety of suitable host cells are available,both eukaryotic and prokaryotic. Examples include bacteria such as E.coli, yeast, filamentous fungi, insect cells, mammalian cells,preferably immortalized, such as mouse, CHO, HeLa, myeloma or Jurkatcell lines, human and monkey cell lines and derivatives thereof. Suchhost cells are useful in drug screening systems to identify agents foruse in diagnosis or treatment of individuals having, or beingsusceptible to corneodesmosin-mediated disease.

The method by which said polynucleotides are introduced into a host cellwill usually depend upon the nature of both the vector/DNA and thetarget cell, and will include those known to a person skilled in theart. Suitable known methods include fusion, conjugation, transfection,transduction, electroporation or injection, as described in Sambrook etal.

In a seventh aspect of the present invention, there is provided atransgenic non-human animal comprising a polynucleotide according to anaforementioned aspect of the invention. Preferably, the transgenic,non-human animal comprises a polynucleotide according to the first orsecond aspects. The transgenic animal may be either homozygous orheterozygous for the variant sequence. The animal, and cells derivedtherefrom, are useful for screening biologically active agents that maymodulate corneodesmosin function. Such screening methods are ofparticular use for determining the specificity and action of potentialtherapies for corneodesmosin-mediated disease, such as psoriasis. Theanimals are useful as a model to investigate the role of corneodesmosinin normal skin function. Transgenic non-human animals are also usefulfor the analysis of the single nucleotide polymorphisms and theirphenotypic effect.

Expression of a polynucleotide of the invention in a transgenicnon-human animal is usually achieved by operably linking thepolynucleotide to a promoter and/or enhancer sequence, preferably toproduce a vector of the fourth aspect, and introducing this into anembryonic stem cell of a host animal by microinjection techniques (Hoganet al. (1986), Manipulating the Mouse Embryo: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, and Capecchi (1989) Science244:1288-1292). Preferably, the construct to be introduced into theanimal additionally comprises (a) a first homology region withsubstantial identity to a first corneodesmosin gene sequence; and (b) asecond homology with substantial identity to a second corneodesmosingene sequence. The first and second homology regions are of sufficientlength for homologous recombination to occur with an endogenouscorneodesmosin gene. Those embryonic stem cells comprising the desiredpolynucleotide sequence may be selected, usually by monitoringexpression of a marker gene, and used to generate a non-human transgenicanimal. Preferred host animals include mice and other rodents. Furtherdevelopment of such an embryonic stem cell may produce a transgenicanimal having cells that are descendant from the embryonic stem cell andthus carry the variant sequence in their genome. Such animals can thenbe selected and bred to produce animals having the variant sequence inall somatic and germ cells. Such mice can then be bred to homozygosity.

In a preferred embodiment, the transgenic non-human animal may comprisean anti sense nucleic acid sequence of the third aspect. The expressionof an anti-sense sequence in a transgenic non-human animal may be usefulin determining the effects of such sequences in treatingcorneodesmosin-mediated disease, or in neutralizing deleterious effectsof variant corneodesmosin genes in an animal. Preferably, the hostanimal will be one which suffers from corneodesmosin-mediated disease.The disease may be naturally occurring or artificially introduced.

In some preferred embodiments, for example where the mediated diseasehas been artificially induced, the transgenic non-human animal will bemodulated to no longer expresses the endogenous corneodesmosin gene.Such animals may be referred to as “knock out.” In some cases, it may beappropriate to modulate the expression of the endogenous corneodesmosingene, or express the polynucleotides of the present invention, inspecific tissues. This approach removes viability problems if theexpression of a gene is abolished or induced in all tissues. Preferably,the specific tissue would be skin. Where the heterologous gene is human,the animal may be useful in identifying agents which inhibit expressionor activity of the variant corneodesmosin sequences of the invention,either in vivo or in vitro.

In an eighth aspect of the present invention there is provided a methodof screening for agents for use in the prognosis, diagnosis or treatmentof individuals having, or being susceptible to, corneodesmosin-mediateddisease, said method comprising contacting a putative agent with apolynucleotide or protein according to an aforementioned aspect of thepresent invention, and monitoring the reaction there between.Preferably, the method further comprises contacting a putative agentwith a reference polynucleotide or protein of FIG. 1 or 2 (SEQ ID NO:1or SEQ ID NO:2, respectively), and comparing the reaction between (i)the agent and the polynucleotide or protein encoding the referenceallele; and (ii) the agent and polynucleotide or protein of theinvention. Potential agents are those which react differently with avariant of the invention and a reference allele. It is envisaged thatthe present method may be carried out by contacting a putative agentwith a host cell or transgenic non-human animal comprising apolynucleotide or proteu1 according to the invention. Putative agentswill include those known to persons skilled in the art, and includechemical or biological compounds, such as anti-sense polynucleotidesequences, complementary to the coding sequences of the first aspect, orpolyclonal or monoclonal antibodies which bind to a product such as aprotein or protein fragment of the second aspect. The agents identifiedin the present method may be useful in determining susceptibility tocorneodesmosin-mediated disease, or in the diagnosis, prognosis ortreatment of said disease.

In a ninth aspect of the present invention, there is provided a methodof diagnosing, or determining susceptibility of a subject tocorneodesmosin-mediated disease, said method comprising determiningwhich allele of one or more of the polymorphisms of the invention ispresent in a subject. The above method maybe used in diagnosing ordetermining susceptibility of a subject to any disease in whichcorneodesmosin is implicated in the pathology, in particularinflammatory disease, such as psoriasis. The method of the ninth aspectmay also be used to identify the presence of a combination of singlenucleotide polymorphisms in a subject which define a haplotype linked tocorneodesmosin-mediated disease. The haplotype may be any particularcombination of the above single nucleotide polymorphisms, optionallyincluding known polymorphisms. Preferred haplotypes are those shown inTable 10a, the most preferred haplotype being B of Table 10a.

Any method, including those known to persons skilled in the art, may beused to determine which allele of one or more polymorphisms of theinvention is present. Preferably, the method comprises first removing asample from a subject. More preferably, the method comprises isolatingfrom a sample a polynucleotide or protein to determine therein whichallele of one or more polymorphisms of the invention is present.

Any biological sample comprising cells containing nucleic acid orprotein is suitable for this purpose. Examples of suitable samplesinclude whole blood, semen, saliva, tears, buccal, skin or hair. Foranalysis of cDNA, mRNA or protein, the sample must come from a tissue inwhich the corneodesmosin gene is expressed, and thus it is preferable touse skin samples.

In a preferred embodiment, the method for diagnosing, or determiningsusceptibility of a subject to a corneodesmosin-mediated disease,comprises determining which allele of one or more polymorphisms of theinvention is present, in a polynucleotide. Any method for determiningalleles in a polynucleotide may be used, including those known topersons skilled in the art. Preferably, the method may comprise the useof anti-sense polynucleotides, as defined above. Such polynucleotidesmay include sequences which are able to distinguish between alleles ofone or more polymorphisms of the invention, by preferential binding, andsequences which hybridize under stringent conditions to a region eitherside of a polymorphism of the invention to enable amplification of oneor more of the polymorphisms.

Methods of this embodiment include those known to persons skilled in theart, for example direct probing, allele specific hybridization, PCRmethodology including Allele Specific Amplification (ASA), and RFLP.

Determination of an allele of a polymorphism using direct probinginvolves the use of anti-sense sequences of the third aspect of theinvention. These may be prepared synthetically or by nick translation.The anti-sense probes may be suitably labeled using, for example, aradiolabel, enzyme label, fluoro-label, biotin-avidin label forsubsequent visualization in, for example, a southern blot procedure. Alabeled probe may be reacted with a sample DNA or RNA, and the areas ofthe DNA or RNA which carry complimentary sequences will hybridize to theprobe, and become labeled themselves. The labeled areas may then bevisualized, for example by autoradiography.

Allele specific amplification (ASA) discriminates between alleles of apolymorphism on the basis of primers which carry 3′ nucleotides specificfor a particular polymorphism. Typically, first and second forwardprimers are provided, wherein the first forward primer hybridizes to oneallele of a polymorphism of the invention, and the second forward primercomprises a mismatch at the polymorphic site, thus preventinghybridization. These primers are used in combination with a backwardprimer, which hybridizes to a distal site to enable amplification of theregion between a forward primer and the backward primer. As the firstforward primer will only bind to a polymorphic site with which itexhibits perfect complementarity, amplification of the region betweenthe forward and backward primers will indicate the presence of aparticular allele. The second forward primer having a mismatch at thepolymorphic site will not hybridize to the particular allele of apolymorphism, and the absence of a amplification product when thisprimer is used indicates the absence of the polymorphism. Preferably,the forward primer will be an anti-sense sequence according to the thirdaspect of the invention. Preferably, the first forward primer willcomprise the complement of a single nucleotide polymorphism of theinvention at the 3′ most position. The backward primer may hybridize toany suitable portion of the corneodesmosin gene to enable amplificationof the intervening region (see, for example, WO 93/22456).

Thus, in a preferred embodiment, there is provided a method fordiagnosing or determining susceptibility of a subject tocorneodesmosin-mediated disease, said method comprising removing asample from a subject and isolating the nucleic acid therefrom;contacting the sample with either a forward primer which preferentiallyhybridizes to one allele of one or more polymorphisms of the presentinvention or a forward primer which comprises a mis-match at thepolymorphic site and does not hybridize thereto, and a backward primerwhich hybridizes to a distal site; subjecting the nucleic acid sample toamplification; and monitoring for presence of an amplification productwhich is indicative of the presence of a particular allele of one ormore of the polymorphisms of the invention. Preferably, a first reactionis performed using one of the forward primers, and a control reaction isthen performed using the other forward primer. It is envisaged that anumber alleles of the single nucleotide polymorphisms of the inventionmay be detected in a single reaction by using multiple primer pairs.Amplification products may then be distinguished by size, usingtechniques known in the art such as gel electrophoresis, or southernblotting. This method allows the unambiguous identification ofindividuals homozygous for either allele as well as heterozygousindividuals.

“RFLP” refers to restriction fragment length polymorphism and is definedas a method of discriminating between two alleles based upon differencesin sequence which result in the presence or absence of a restrictionenzyme recognition site. In a preferred embodiment of the present aspectthere is provided a method for diagnosing or determining susceptibilityto corneodesmosin-mediated disease, said method comprising removing anucleic acid sample from a subject, and contacting with one or moreappropriate restriction enzymes. The size of fragments produced isindicative of which allele of one or more single nucleotide polymorphismaccording to the invention is present. An allele of a polymorphism ofthe invention may naturally produce a restriction enzyme site, thusallowing for determination of its presence by analysis of therestriction fragments produced. In some cases, however, an allele of apolymorphism does not create a restriction enzyme site, and one must beartificially introduced. This may be done by using a suitable mis-matchprimer, according to methods known in the art.

The appropriate restriction enzyme will, of course, be dependent uponthe polymorphism and restriction site, and will include those known topersons skilled in the art. Preferred restriction enzymes are listed inTable 3 (ii), Column 8, with the expected fragment sizes in Columns 9,10, and 11. Analysis of the digested fragments may be performed usingany method in the art, for example gel analysis, or southern blots.

Preferably, the method may first comprise the amplification of a regionof the corneodesmosin gene containing one or more of the polymorphicsites of the invention, for example, using PCR techniques. The probes ofthe present invention may be useful for this purpose.

The above-described methods may require amplification of the DNA samplefrom the subject, and this can be done by techniques known in the art,such as PCR (see Erlich (ed.) (1992) PCR Technology: Principles andApplications for DNA Amplification Freeman Press, NY; Innis et al.(1990) PCR Protocols: A Guide to Methods and Applications, AcademicPress, San Diego, Calif.; Mattila et al. (1991) Nuc. Acids Res. 19:4967;Eckert et al. (1991) PCR Methods and Applications 1:17; and U.S. Pat.No. 4,683,202). Other suitable amplification methods include ligasechain reaction (LCR) (Wu et al. (1989) Genomics 4:560; Landegran et al.(1988) Science 241:1077); transcription amplification (Kwoh et al.(1989) PNAS 86:1173); self-sustained sequence replication (Guatelli etal. (1990) PNAS 87:1874) and nucleic acid-based sequence amplification(NASBA). The latter two methods both involve isothermal reactions basedon isothermal transcription which produce both single stranded RNA anddouble-stranded DNA as the amplification products, in a ratio of 30 or100 to 1, respectively.

It may often be desirable to identify the presence of multiple singlenucleotide polymorphisms in a sample from a subject. This may be thecase in the present invention where the corneodesmosin gene contains 39polymorphisms, each of which may be indicative of a different phenotype.For this purpose, nucleic acid arrays may be useful, as described in WO95/11995. The array may contain a number of probes, each designed toidentify one or more of the above single nucleotide polymorphisms of thecorneodesmosin gene, as described in WO 95/11995.

In a further preferred embodiment of the ninth aspect, the method maycomprise determining which allele of one or more polymorphisms ispresent in a protein of the invention Any method for determining thepresence of a particular form, or allele, of a protein is present, maybe used. One such method involves the use of antibodies in diagnosing ordetermining susceptibility to corneodesmosin-mediated disease. Themethod may comprise removing a sample from a subject, contacting thesample with an antibody to an antigen of a protein or protein fragmentsaccording to the second aspect of the present invention, and detectingbinding of the antibody to the antigen, wherein binding is indicative ofthe presence of a particular allele or form of the protein and thus riskto corneodesmosin-mediated disease. Tissue samples as described aboveare suitable for this method.

The detection of binding of the antibody to the antigen in a sample maybe assisted by methods known in the art, such as the, use of a secondaryantibody which binds to the first antibody, or a ligand. Immunoassaysincluding immunofluorescence assays (IFA) and enzyme linkedimmunosorbent assays (ELISA) and immunoblotting may be used to detectthe presence of the antigen. For example, where ELISA is used, themethod may comprise binding the antibody to a substrate, contacting thebound antibody with the sample containing the antigen, contacting theabove with a second antibody bound to a detectable moiety (typically anenzyme such as horse radish peroxidase or alkaline phosphatase),contacting the above with a substrate for the enzyme, and finallyobserving the color change which is indicative of the presence of theantigen in the sample.

In a tenth aspect of the invention, there is provided a method oftreating a subject who has been diagnosed as having, or beingsusceptible to, corneodesmosin-mediated disease such as psoriasis. Themode of treatment will depend upon the nature of the polymorphism(s) andthe phenotypic effect, and preferably comprises negating the effect ofthe disease causing polymorphism(s). Where a subject has been diagnosedaccording to the methods of the invention, treatment to negate theeffect of the disease causing polymorphism may include any suitablemeans. A suitable treatment includes the administration of apolynucleotide sequence which hybridizes, preferably under stringentconditions (as defined above) to the corneodesmosin gene. Suchpolynucleotide sequences may include the anti-sense sequences of thethird aspect. Alternatively, the treatment may comprise a polynucleotidesequence encoding the corneodesmosin gene or a fragment thereof, andhaving either a reference or variant allele of a polymorphism of theinvention. Preferably, the method comprises (i) determining which alleleof one or more polymorphisms of the invention are present; and (ii)administering a polynucleotide sequence which hybridizes under stringentconditions to the corneodesmosin gene, or a polynucleotide sequenceencoding the reference sequence of the corneodesmosin gene or a fragmentthereof, or a polynucleotide sequence of the first aspect.

In an alternative embodiment of this aspect, there is provided the useof a polynucleotide sequence of the tenth aspect in the manufacture of amedicament for use in the diagnosis and treatment ofcorneodesmosin-mediated disease.

This method of diagnosis and treatment may comprise determining andintroducing alleles in the form of a polynucleotide or protein. In theabove embodiments, the allele of a polymorphism may be determined usingany method, as discussed above. The treatment may be introduced in theform of a protein or polynucleotide. Any suitable means for introductionof a protein may be used. Introduction of a polynucleotide may use genetherapy methods including those known in the art. In general, apolynucleotide encoding the allele will be introduced into the targetcells of a subject, usually in the form of a vector and preferably inthe form of a pharmaceutically acceptable carrier. Any suitable deliveryvehicle may be used, including viral vectors, such as retroviral vectorsystems which can package a recombinant genome. The retrovirus couldthen be used to infect and deliver the polynucleotide to the targetcells. Other delivery techniques are also widely available, includingthe use of adenoviral vectors, adeno-associated vectors, lentiviralvectors, pseudotyped retroviral vectors, and pox or vaccinia virusvectors. Liposomes may also be used, including commercially availableliposome preparations such as Lipofectin®, Lipofectamine®, (GIBCO-BRL,Inc., Gaithersburg, Md.), SuperFect® (Qiagen GmbH, Hilden, Germany), andTransfectam® (Promega Biotec Inc., Madison, Wis.).

The polynucleotide or vehicle may be administered parenterally (e.g.,intravenously), transdermally, by intramuscular injection, topically, orthe like. As corneodesmosin-mediated diseases are usually manifested inthe skin, topical administration is preferred. The exact amount ofpolynucleotide or vehicle to be administered will vary from subject tosubject and will depend upon age, weight, general condition, andseverity or mechanism of the disorder.

In a further aspect, the present invention provides a kit for thedetection in a subject of a single nucleotide polymorphism according tothe present invention. Preferably, the kit will contain polynucleotidesaccording to the aforementioned aspects, most preferably the anti-sensesequences of the third aspect for use as probes or primers; antibodiesof the fifth aspect; or restriction enzymes for use in detecting thepresence of a polynucleotide, protein, or protein fragment of theinvention. Preferably, the kit will also comprise means for detection ofa reaction, such as nucleotide label detection means, labelled secondaryantibodies or size detection means. In yet a further preferredembodiment, the polynucleotides, or antibodies may be fixed to asubstrate, for example an array, as described in WO 95/11995.

The preferred embodiments of each aspect apply to the other aspects ofthe invention, mutatis mutandis.

The present invention will now be described by way of a non-limitingexample with reference to the following figures in which:

FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) of the genomic cloneof the corneodesmosin gene, of GenBank Accession No. AC006163;

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) of the corneodesmosinprotein and coding sequence therefor; and

FIG. 3 shows the exon and intron structure of the corneodesmosin gene.

EXAMPLES Determination of Gene Structure

The mRNA sequence of the corneodesmosin gene (GenBank Accession IDNM_(—)001264) was used to screen the following public DNA databases:(available through the National Centre for Biotechnology Information Website—www.ncbi.nlm.nih.gov); NR (Non-Redundant DNA), HTGS (HighThroughput Genomic Sequence), and GSS (Genome Survey Sequence). Theanalysis was performed using the BLASTN algorithm (Altschul et al.(1990) J. Mol. Biol. 215:403-410). Any genomic sequences containing thecorneodesmosin gene were identified by their degree of sequenceidentity. The gene structure was determined by comparison of the mRNAsequence with the genomic clones, The deduced exon-intron organisationof the corneodesmosin gene is presented in FIG. 3.

Oligonucleotide Primer Design—for Corneodesmosin Gene Sequencing

Five pairs of oligonucleotide primers (S1F/S1R; S2.1F/S2.1R;S2.2F/S2.2R; S2.3F/S2.3R; S2.4F/S2.4R, S2.5F/S2.5R—Table 1) weredesigned to amplify exons 1 and 2 of the corneodesmosin gene including350 bp 5′ untranslated region (UTR) and 909 bp 3′ UTR sequences.Oligonucleotide primer sequences were derived from human chromosome 6p21genomic DNA sequence (GenBank Accession AC006163).

TABLE 1 Oligonucleotide Primer DNA Sequences Primer ID Primer SequenceSEQ ID NO S 1F dCTGGGTCCCGTGGCAAGA 5 S 1R dGTCCTCTCCCGGAGTCTC 6 S 2.1FdGGTGAGGGAGGAAGCCAAG 7 S 2.1R dGAGCTGACGCTTTGGCCAC 8 S 2.2FdGCCAACCAATGACAACTCTTACC 9 S 2.2R dGCCTCCACAGAGCTGGAC 10 S 2.3FdGGCAAATACTTCTCCAGCAACC 11 S 2.3R dGGCCTTCTCCCATATGGGA 12 S 2.4FdCCAAGGAGAGTTACTCGACAG 13 S 2.4R dGGCATATTGGGTGGGTTGAC 14 S 2.5FdCATCTGGAAACAGTGGCCAC 15 S 2.5R dGTCTTCCTCCTCTGTGGGAG 16

Corneodesmosin Gene Amplification

Genomic DNA from a panel of 24 unrelated individuals was amplified usingprimer pairs S1F/S1R; S2.1F/S2.1R; S2.2F/S2.2R; S2.3F/S2.3R;S2.4F/S2.4R, S2.5F/S2.5R. 100 ng genomic DNA was amplified by PCR in atotal reaction volume of 25 μl containing 50 mM KCl, 20 mM Tris-HCl (pH8.4), 2 mM MgCl₂ 200 μM each dATP, dCTP, dGTP, dTTP, 1 μM eacholigonucleotide primer and 0.5 units AmpliTaq Gold DNA polymerase(Applied Biosystems). Reactions were thermocycled with an initialdenaturation step of 95° C./10 min followed by 35 cycles of 94° C./30sec; T_(m) annealing/30 sec; 72° C./30 sec. A final elongation step of72° C./10 min completed the amplification. Annealing temperatures(T_(m)) for specific primer pairs are presented in Table 2.

TABLE 2 Primer Annealing Temperatures and Amplimer Sizes Fragment sizeTm Amplimer Primer Pairs (bp) (° C.) 1 S1F and S1R 495 63 2.1 S2.1F and610 62 S2.1R 2.2 S2.2F and 619 62 S2.2R 2.3 S2.3F and 621 63 S2.3R 2.4S2.4F and 532 59 S2.4R 2.5 S2.5F and 474 61 S2.5R

Heteroduplex Analysis Using DHPLC

Oligos were designed to amplify products of between 400-800 bp in lengthfrom the genomic DNA of 12-25 individuals. Denaturing high-performanceliquid chromatography (DHPLC) analysis was performed using the WAVE™ DNAfragment analysis system (Transgenomic) (Kuklin et al. (1997-98) Genet.Test. 1(3):201-206). The temperature required for successful resolutionof heteroduplex molecules within each PCR product was determinedempirically by injecting PCR product at a series of increasing mobilephase temperatures and constructing a fragment specific melting curve. Auniversal gradient for double-stranded DNA was used to determine theappropriate acetonitrile concentration for the heteroduplexidentification. For mutation detection, 1-2 ul aliquots of the PCRreactions from each of the eleven individuals were injected onto theWAVE™ column. Mutation detection gradients were for four minutes.Results were graphically visualized using the D-7000 HSM software(Transgenomic).

Direct Sequencing of PCR Products

50-100 ng of PCR products were sequenced in both orientations using theDYEnamic ET terminator cycle sequencing premix kit from Amersham.Reactions were fractionated on ABI 377 automated sequencers usingstandard procedures. Chromatographic traces were analyzed using theSEQUENCHER program (Gene Codes, USA), to identify SNP positions.

Detection of Variant Alleles—Assay Design for Genotyping

The fragment sequence containing the polymorphism was analyzed for thecreation or deletion of a naturally occurring restriction enzymerecognition site in response to variation in the nucleotide sequence. Ifthe polymorphism did not result in any changes in restriction enzymerecognition sites, then the sequence was interrogated with the PrimerDesign Mismatch Program™. This is an adaptation of the program describedby Davidow (1992) Comput. Appl. Biosci. 8:193-194.

Detection of Polymorphisms in 24 Population Controls

The application of the approach outlined above resulted in theidentification of 39 SNPs. These are described in Table 3, in which:

-   -   Column 1 designates each single nucleotide polymorphism a        reference number.    -   Column 2 provides the positional reference of the polymorphism        with respect to FIG. 1, together with details of the        polymorphism itself. For example, the reference “C6948T”        indicates a substitution of the nucleotide “C” for nucleotide        “T” at position 6984 of FIG. 1.    -   Column 3 of (i) provides the corresponding positional references        with respect to the coding sequence of the corneodesmosin gene.    -   Column 4 of (i) indicates the region of the gene which the        polymorphism occurs.    -   Column 5 of (i) shows the sequence flanking the polymorphism,        the polymorphism itself being shown in bold type. The single        nucleotide polymorphisms are defined using standard IUB code.    -   Column 6 of (i) indicates the SEQ ID NO of the corresponding        flanking sequence.    -   Columns 3 and 5 of (ii) show primer sequences which may be used        to amplify a region of the corneodesmosin gene to enable        detection of the single nucleotide polymorphism by using        restriction enzyme analysis. The amplified product size is shown        in Column 7 of (ii).    -   Columns 4 and 6 of (ii) indicate the SEQ ID NO of the        corresponding primer sequence.    -   Columns 8 to 10 of (ii) list the restriction enzymes used to        digest the amplified product, and the sizes of fragments        generated by the reference, variant and heterozygous sequences        respectively.

RFLP or ASA assays were developed for all of these SNPs, and thecorresponding primers along with amplification product and digestionfragment sizes are also given in Table 3. Of these 39 SNPs, 9 give riseto amino acid changes. These are shown in Table 4.

Additional Corneodesmosin Polymorphisms

In a subsequent experiment, DNAs from 96 individuals comprising 24 typeIA psoriatics, 24 type IB psoriatics, 24 type II psoriatics, and anadditional 24 healthy controls were sequenced as described above usingprimers designed to cover the remainder of the corneodesmosin gene (seeTable 5a)

The sequencing reactions were carried out with 50-100 ng of PCR productssequenced in both orientations using the DYEnamic ET terminator cyclesequencing premix kit from Amersham according to the following protocol.

The PCR products were Exo/Sap treated and desalted using p10 columns,prior to setting up the sequencing reactions in a thermowell plateincluding:

200-400 ng PCR Product

1 μl primer@10 pmol ml⁻¹

8 μl ET Termination mix

H₂O to 20 μl

The plates were sealed with an MJ Research Microseal film and thenvortexed to mix samples, followed by a spin to ensure reaction is at thebottom of the wells.

PCR was carried out according to the following protocol:

-   -   No Predenaturation    -   95° C. for 30 sec    -   50° C. for 15 sec    -   60° C. for 1 min    -   for 40 cycles and then hold at 10° C. until ready to purify.

After removing the plate from the thermocycler, the products werepurified by ethanol precipitation. To each well we added 2 μl 7.5Mammonium acetate followed by 80 μl 100% ethanol, and incubated at roomtemperature for 10 minutes before spinning at 4000 rpm for 1 hour atroom temperature. The supernatant was discarded and the pellet washedwith 70% ethanol before centrifugation for a further 30 minutes. Thesupernatant was discarded and remaining ethanol removed gently bypipetting using p10 tips before allowing the pellets to air dry.

The samples were then resuspended in 10 μl MegaBACE Loading Buffer(Molecular Dynamics) and transferred to a Robbins plate prior to loadingonto the MegaBACE. Reactions were fractionated on a Molecular DynamicsMegaBACE capillary sequencer using standard procedures. Chromatographictraces were analysed using the SEQUENCHER programme (Gene Codes, USA) toidentify SNP positions.

A total of 28 novel SNPs were identified (additional to those given inthe example above). For reference, these are SNPs 6-18 and 53-67 inTable 5b. A combined list of corneodesmosin SNPs is given in Table 6.

Corneodesmosin Gene Association with Psoriasis

A total of 21 SNPs (see Table 7) were genotyped in 147 familiesidentified through a proband with psoriasis (a total of 499 individuals,of whom 233 were affected). The genotyping was carried out using avariety of methods (single base extension using the Snapshot kit fromAmersham Pharmacia Biotech, Pyrosequencing (Ahmadian et al. (2000) Anal.Biochem. 280:103-110) or direct sequencing as given in Table 7. Allthese methods used established methodologies that are provided by theequipment manufacturers and/or are well known to those skilled in theart.

Linkage Disequilibrium

The extent of linkage disequilibrium (LD) between markers was calculatedusing genotype data from 199 unrelated, unaffected individuals and isexpressed as correlation coefficients in Table 8. This analysis showsthat there is extensive linkage disequilibrium between many of thecorneodesmosin polymorphisms.

Single Point Association

Single point associations between each SNP and psoriasis affected statuswere calculated using the TRANSMIT program (D. Clayton, MRCBiostatistics Unit, Cambridge)—see Table 9. Highly significantassociations were observed between SNPs 19, 21, 23, 24, 26, 28, 30, 33234, 37, 38, and psoriasis. The single SNP showing the most significantassociation with psoriasis that has been previously reported is SNP 33(Tazi-Ahnini et al. (1999) Hum. Mol. Genet. 8:1135-1140; Allen et al.(1999) Lancet 353:1589-1590).

This study has identified nine SNPs, (19, 21, 24, 26, 28, 30, 34, 37,and 38) which show global chi-squared values greater than that seen forSNP 33, and are therefore more powerfully predictive of affected status.

Haplotype Analysis

A total of 19 SNPs were used for haplotype analysis (SNPs at positions29 and 32 were excluded due to low information content). Three commonhaplotypes were identified (Table 10). Of the three common haplotypes,haplotype B is significantly associated with psoriasis. The alleles arecoded alphabetically (Table 10b) such that the nucleotide first in thealphabet is coded as 1, and the other nucleotide is coded as 2. Thus, Ais always 1, T is always 2, and G or C are coded depending on the othernucleotide. For example, in SNP No. 1, which is a C to T substitution,the presence of the C allele is coded as 1 and the presence of the Tallele is coded as 2 (see Table 10b). In Table 10a, this means thathaplotypes A and B have C residues, and haplotype C has a T residue atthis position. For an A to C substitution, the A allele will be coded as1, and the C allele as 2. In a C to G substitution, the C allele will becoded as 1 and the G allele as 2.

Construction of Corneodesmosin Gene Targeting Vector

As the genetic data pointed strongly to an involvement of thecorneodesmosin gene in the pathophysiology of psoriasis, we decided toengineer mouse strains in which the mouse orthologue of thecorneodesmosin gene is knocked out by homologous recombination using avector construct designed to remove exon 2 of the corneodesmosin gene.

Murine corneodesmosin genomic clones were isolated from a mouse largeinsert PAC library, using mouse corneodesmosin cDNA sequence as a probeby standard techniques. The isolated murine corneodesmosin genomicclones were then restriction mapped in the region of the corneodesmosingene using small oligonucleotide probes and standard techniques. Themurine genomic locus was partially sequenced to enable the design ofhomologous arms to clone into the targeting vector. The murinecorneodesmosin gene is a two-exon gene. A 4 kb 5′ homologous arm and a 1kb 3′ homologous arm where amplified by PCR and the fragment cloned intothe targeting vector. The position of these arms was chosen tofunctionally disrupt the corneodesmosin gene by deleting the majority ofthe coding sequence. A targeting vector was prepared where the deletedcorneodesmosin sequence was replaced with non-homologous sequencescomposed of an endogenous gene expression reporter (an in frame fusionwith lacZ) upstream of a selection cassette composed of a self promotedneomycin phosphotransferase (neo) gene in the same orientation as thecorneodesmosin gene.

Transfection and Analysis of Embryonal Stem Cells

Embryonal stem cells (Evans and Kaufman (1981) Nature 292:154-156) werecultured on a neomycin-resistant embryonal fibroblast feeder layer grownin Dulbecco's Modified Eagles medium supplemented with 20% Fetal CalfSerum, 10% new-born calf serum, 2 mM glutamine, non-essential aminoacids, 100 μM 2-mercaptoethanol and 500 u/ml leukemia inhibitory factor.Medium was changed daily and ES cells were subcultured every three days.5×10⁶ ES cells were transfected with 5 μg of linearized plasmid byelectroporation (25 μF capacitance and 400 Volts). 24 hours followingelectroporation the transfected cells were cultured for nine days inmedium containing 200 μg/ml neomycin. Clones were picked into 96 wellplates, replicated and expanded before being screened by PCR to identifyclones in which homologous recombination had occurred between theendogenous corneodesmosin gene and the targeting construct. From 96picked clones, 45 targets were identified. These clones where expandedto allow replicas to be frozen and sufficient high quality DNA to beprepared for Southern blot confirmation of the targeting event usingexternal 5′ and 3′ probes, all using standard procedures (Russ et al.(2000) Nature 404:95-99).

Generation of Corneodesmosin-Deficient Mice

C57BL/6 female and male mice were mated and blastocysts were isolated at3.5 days of gestation. 10-12 cells from Clone 7 (described in Example 2)were injected per blastocyst and 7-8 blastocysts were implanted in theuterus of a pseudopregnant F1 female. Five chimeric pups were born ofwhich one male was 100% agouti (indicating cells descendent from thetargeted clone). This male chimera was mated with female and MF1 and 129mice, and germline transmission was determined by the agouti coat colorand by PCR genotyping, respectively.

Corneodesmosin Knock-Out Mouse as a Model of Corneodesmosin-MediatedDisease

Mice heterozygous for the corneodesmosin knockout are superficiallynormal. Staining for expression of the lacZ reporter gene fused to thecorneodesmosin promoter in the knockout construct shows clear expressionin desquamating skin. We then genotyped surviving offspring fromintercrosses of heterozygous knockout mice on an outbred geneticbackground in an attempt to isolate mice homozygous for the knockout.

From 44 surviving progeny we identified:

-   -   17 wild type    -   27 heterozygotes    -   0 homozygous mutant.

Statistical analysis of these data indicate that the ratio of wildtype:heterozygous animals conforms to a 1:2 ratio consistent with ahomozygous lethal phenotype (Chi square=0.557).

In keeping with this analysis, two pups found dead 24-48 hours afterbirth were homozygous mutant. Together, these data indicate thecorneodesmosin deficiency in mice is lethal with pups dying soon afterbirth, most likely through dehydration as a result of failure toestablish a permeability barrier in the skin.

We conclude from this that altering the activity of corneodesmosin(e.g., by modulating expression or altering its proteolytic processing)will be useful in developing models of disease in which epithelialintegrity is increased (e.g., psoriasis) or decreased (e.g.,dermatitis), and for testing novel agents for the alleviation ofcorneodesmosin-mediated disease.

TABLE 3i S Gene SNPs With Location and Assay Details Corneo- desmosinSEQ SNP nt ID SNP nt position position Location Flanking Sequence NO 1C6984T −115 5′ UTR CTCCCGGCCA CACCAACTTC CCCCYGGGCA CCCACCCCCTCCACCTCTCC 17 2 A7068G −31 5′ UTR AATGTCCAGCTCTGGCATAA AGGACCCRGGTGTCCTCGAG CTGCCATCAG 18 3 C7077T −22 5′ UTR TCTGGCATAA AGGACCCAGGTGTCCTYGAG CTGCCATCAG TCAGGAGGCC 19 4 C7107T 9 5′ UTR CTGCCATCAGTCAGGAGGCCGTGCAGYCCG AGATGGGCTC GTCTCGGGCA 20 5 A7164T 66 CodingSequence GGCGTGTGGGTGGGCACGGG ATGWTGGCAC TGCTGCTGGC TGGTCTCCTC 21 6C10039T 137 Coding Sequence CTAAGAGCAT TGGCACCTTC TCAGACCCYTGTAAGGACCCCACGCGTATC 22 7 C10082T 180 Coding SequenceACCTCCCCTAACGACCCCTGCYTCACTGGGAAGGGTG 23 8 C10134T 206 Coding SequenceCAGTAGCTAC AGTGGCTCCA GCAYTTCTGG CAGCTCCATTTCCAGTGCCA 24 9 G10344A 442Coding Sequence GAGCAGCAGC TCTCACTCGG GAARCAGCGGCTCTCACTCG GGAAGCAGCA 2510 10363(AAG)ins 461 Coding Sequence GAAGCAGCGGCTCTCACTCG GG(AAG)CAGCAGCTCTCATTCGAGCAGCAGC 26 11 A10516G 614 Coding SequenceCTGGACAAAGCTCTTCCTCT TCCCARACCT CTGGGGTATC CAGCAGTGGC 27 12 C10521T 619Coding Sequence CTGGACAAAGCTCTTCCTCT TCCCAAACCT YTGGGGTATC CAGCAGTGGC 2813 T10624C 722 Coding Sequence GGAGGGCCCA TCGTCTCGCA CTCYGGCCCCTACATCCCCA GCTCCCACTC 29 14 G10669A 767 Coding SequenceGCTCCCACTCTGTGTCAGGG GGTCAGAGRC CTGTGGTGGT GGTGGTGGAC 30 15 T10873C 971Coding Sequence CCTACAGTAA GGGTAAAATC TAYCCTGTGG GCTACTTCAC CAAAGAGAAC31 16 G11020A 1118 Coding Sequence AGCCAGTCGGCAGCTTCCTC GGCCATTGCRTTCCAGCCAG TGGGGACTGG 32 17 A11117G 1215 Coding SequenceCTCCCTCCAGTTCTCGAGTC CCCAGCRGTT CTAGCATTTC CAGCAGCTCC 33 18 T11138G 1236Coding Sequence CCCAGCAGTT CTAGCATTTC CAGCAGCKCC GGTTCACCCTACCATCCCTG 3419 G11142T 1240 Coding Sequence CTAGCATTTC CAGCAGCTCCGKTTCACCCTACCATCCCTGCGGCAGTGCT 35 20 C11145T 1243 Coding SequenceCTAGCATTTC CAGCAGCTCC GGTTYACCCTACCATCCCTGCGGCAGTGCT 36 21 G11233C 1331Coding Sequence GCAGCAGCTC CAGTTCCCAA TCSAGTGGCA AAATCATCCTTCAGCCTTGT 3722 T11260C 1358 Coding Sequence TCGAGTGGCA AAATCATCCTTCAGCCTTGYGGCAGCAAGT CCAGCTCTTC 38 23 G11495A 1593 Coding Sequence TTCCTACCCCAAGGAGAGTT ACTCRACAGTCCATAAGTCA ACTGTTGTGT 39 24 11505(AAG)ins 16033′ UTR GAGAGTTACTCGACAGTCCATAAG(AAG)TCAACTGTTGTGTGTGTGCATGC 40 25G11576T 1674 3′ UTR TACACTATATCCCATATGGGAGAAGKCCAGTGCCCAGGCATAGGGTTAGC41 26 T11641C 1739 3′ UTRCCCAAAAGAGTGGTTCTGCTTTCTCYACTACCCTAAGGTTGCAGACTCTC 42 27 T11649C 17473′ UTR AGTGGTTCTGCTTTCTCTACTACCCYAAGGTTGCAGACTCTCTCTTATCA 43 28 T11808G1906 3′ UTR CCCCTTACAATTCCCTCTACTGTGTKGAAATGGTCCATTGAGTAACACCC 44 29C11839G 1937 3′ UTR GGTCCATTGAGTAACACCCCCATCASCTTCTCAACTGGGAAACCCCTGAA45 30 C11885T 1983 3′ UTRTGAAATGCTCTCAGAGCACCTCTGAYGCCTGAAGAAGTTATACCTTCCTC 46 31 C11977T 20753′ UTR AAACAGTGGC CACTTTTCAC TGACCTYTCT TCGACATCTA GTCAACCCAC 47 32T12018C 2116 3′ UTR CAACCCACCCAATATGCCACTGGGCYTTCGCTCCCAATTCCACCCCACCC48 33 T12136C 2234 3′ UTRTTATCTCAGCCCCTTCCTGTGGCCAYTTCCCTCAGTGCCCAGATGATTCC 49 34 C12149T 22473′ UTR TTCCTGTGGCCATTTCCCTCAGTGCYCAGATGATTCCCTGGGTGAGGGAG 50 35 G12198A2296 3′ UTR GACACTGGGGCACCCTCAGAGGTTGRAGCAGGCTCCCTGCTGTCCCTGGA 51 36G12283A 2381 3′ UTR GGTGCAGACTTTTTGCCTTCTTGGARTCCTGGGTCTCCTCTGAGAGTCTG52 37 T12318C 2416 3′ UTRTCCTCTGAGAGTCTGGGTGGTGCTCYTCCTACGCCTCTAGAGGTCTCTGT 53 38 C12345T 24433′ UTR CCTACGCCTCTAGAGGTCTCTGTGTYCCTCATTTTCCTTCAAAAGCGGGC 54 39 G12373A2471 3′ UTR TCATTTTCCTTCAAAAGCGGGCTGTRTTTCTCTTCTACCTTCCAGCTCCT 55

TABLE 3ii PCR SNP SEQ SEQ product nt ID ID size Allele Allele Hetero-SNP position Primer sequence NO Primer sequence NO (bp) Enzyme 1 2zygote 1 C6984T dCTGGGTCCCGTGGCAAGA 5 dGTCCTCTCCCGGAGTCTC 6 496 Ava I313, 220, 313, 220, 32, 16, 93, 32, 135, 93, 135 16, 135 32 16 2 A7068GdCTGGGTCCCGTGGCAAGA 5 dCTGACTGATGGCAGCTCGAG 58 333 Pvu II 333 309, 24333, 309, GACAGC 24 3 C7077T dCTGGGTCCCGTGGCAAGA 5 dGTCCTCTCCCGGAGTCTC 6496 Tag I 496 315, 496, 315, 181 181 4 C7107T dCCCACCCCCTCCACCTCT 59dCCGTCCCCTTCGCTGGGTCC 60 283 Ava I 150, 150, 150, 85, TC 85, 48 85, 32,48, 32, 16 16 5 A7164T dATTACCACGCTCCTCCCG 61 dGCAGGAGGAGACCAGCCAGC 62249 Hinc 249 220, 29 249, 220, AGCAGTGTCA II 29 6 C10039TdCAGTTCTTCCTCCTTTCTCCA 63 dAGGGGAGGTGATACGCGTGG 64 215 BstN I 215 184,31 215, 184, T GGTCCTTCCA 31 7 C10082T dGACCTTGGCTAAGAGCATTG 65dCCTGGCTTAAAAGATCCTGC 66 240 Mnl I 240 151, 240, 151, 70, 19 70, 19 8C10134T dGGTGAGGGAGGAAGCCAAG 7 dAGAACTGCTGGAGCCACTGT 68 193 Pst I 193163, 30 193, 163, AGCTACTGCA 30 9 G10344A dCAGCTGGGGAGCAGCAGCTCT 69dGAGCTGACGCTTTGGCCAC 8 243 Bsl I 243 219, 22 243, 219, CCCTCGGGA 22 1010363 dAGCGGCTCTCACTCGGGAAG 71 dTGACGCTTTGGCCACTGCTG 72 204 204 204 204(AAG)ins dAGCGGCTCTCACTCGGGCAG 73 11 A10516G dCAGCCTGGACAAAGCTCTTCC 74dCTGGAAGGCCACCATTGCTA 75 269 Dde I 269 243, 26 269, 243, TCTTCTCA 26 12C10521T dGCCAACCAATGACAACTCTTA 9 dGCCTCCACAGAGCTGGAC 10 620 BsrF I 162132, 30 162, 132, CC 30 13 T10624C dCTGCAGTGGAGGGCCCATCGT 78dCTGGAAGGCCACCATTGCTA 79 162 BsmA 451, 401, 451, 401, CTCGCACAC I 169169, 50 169, 50 14 G10669A dGCCAACCAATGACAACTCTTA 76 dGCCTCCACAGAGCTGGAC77 620 Mbo I 190, 221 221, 190, CC 31 31 15 T10873CdAGGCATGACCTACAGTAAGGG 80 dGCCTCCACAGAGCTGGAC 77 221 Mnl I 62, 62, 12,270, 258, TAAAATCGA 270, 258, 186, 62, 45, 45, 57, 45, 186, 186, 57 1257 16 G11020A dCAGCCAGTCGGCAGCTTCCTC 81 dTGAAGGAGCCGGTGCCTG 82 225 BstUI 225 195, 30 225, 195, GGCCATCGC 30 17 A11117G dTGCTCTCCCTCCAGTTCTCGA83 dGTGTCAAGGAGGAGACAGAC 84 231 Pst I 231 199, 32 231, 199, GTCCCCTGC A32 18 T11138G dGGCAAATACTTCTCCAGCAAC 11 dGGCCTTCTCCCATATGGGA 12 622 HhaI 622 440, 622, 440, C 182 182 19 G11142T dGGCAAATACTTCTCCAGCAAC 11dGGCCTTCTCCCATATGGGA 12 622 Msp I 241, 181, 241, 214, C 214, 60, 181,167, 167 214, 60 167 20 C11145T dGTTCTAGCATTTCCAGCAGCT 87dGTGTCAAGGAGGAGACAGAC 88 200 Hinf I 200 176, 24 200, 176, CCGATT A 24 21G11233C dGGCAAATACTTCTCCAGCAAC 11 dGGCCTTCTCCCATATGGGA 12 622 Taq I 146,146, 389, 262, C 389, 127, 146, 127, 87 262, 87 87 22 T11260CdGGCAAATACTTCTCCAGCAAC 11 dGTGACCAGAAGAGCTGGACT 89 331 Hha I 331 304, 27331, 304, C TGCTGGC 27 23 G11495A dGGCAAATACTTCTCCAGCAAC 11dGGCCTTCTCCCATATGGGA 12 622 Taq I 146, 146, 349, 262, C 127, 127, 146,127, 349 262, 87 87 24 11505 dGGAGAGTTACTCGACAGTCCA 90dCAGTAGGAGAGAATCAAGAG 91 259 259 259 259 (AAG)ins TAAGAAG AGGAGCdGGAGAGTTACTCGACAGTCCA 92 dCAGTAGGAGAGAATCAAGAG 91 TAAGTCA AGGAGC 25G11576T dAAGGAGAGTTACTCGACAGTC 93 dAGGAGAGAATCAAGAGAGGA 94 254 Hae 25496, 158 254, 158, C GC III 96 26 T11641C dAAGGAGAGTTACTCGACAGTC 93dTAAGAGAGAGTCTGCAACCT 95 190 Aci I 190 160, 30 190, 160, C TAGGGTAGC 3027 T11649C dAAGGAGAGTTACTCGACAGTC 93 dAGGAGAGAATCAAGAGAGGA 94 254 Bsu254 168, 86 254, 168, C GC 36 I 86 28 T11808G dAGGTTGCAGACTCTCTCTTAT 96dATGGGGGTGTTACTCAATGG 97 186 Hinc 186 158, 28 186, 158, CACCC ACCATGTCII 28 29 C11839G dAGGTTGCAGACTCTCTCTTAT 96 dAAGTGGCCACTGTTTCCAGA 98 315Alu I 315 252, 63 315, 252, CACCC TGATGG 63 30 C11885TdAGGTTGCAGACTCTCTCTTAT 96 dAAGTGGCCACTGTTTCCAGA 98 315 BsaH 315 234, 81315, 234, CACCC TGATGG I 81 31 C11977T dCCATCATCTGGAAACAGTGG 99dCGTGGTGAGCTCTGTAATGG 100 124 Ear I 124 80, 44 124, 80, 44 32 T12018CdACCATCATCTGGAAACAGTGG 101 dTGAGCTCTGTAATGGAGGGT 102 120 Ban II 120 80,38, 120, 80, C GG 2 38, 2 33 T12136C dCCTTATCTCAGCCCCTTCCTG 103dATCTGTCCAGGATCCAGGGA 104 126 Ear I 126 94, 32 126, 94, TGGCCT CAGC 3234 C12149T dAACACACCCATTGCCTCTCAA 105 dCCACAGTTTACTGAGCCATC 106 167 Bsi167 102, 65 167, 102, G TG HKA I 65 35 G12198A dCCTTATCTCAGCCCCTTCCTG107 dAGGATCCAGGGACAGCAGGG 108 118 Sau 118 89, 29 118, 89, TGGC AGCCTGGT96 I 29 36 G12283A dGGACAGATGGCTCAGTAAACT 109 dAGGGACACAGAGACCTCTAG 110122 Tfi I 122 67, 55 122, 67, G 55 37 T12318C dGGACAGATGGCTCAGTAAACT 109dAGGGACACAGAGACCTCTAG 110 122 Ear I 122 95, 27 122, 95, G 27 38 C12345TdCTCTTCCTACGCCTCTAGAGG 111 dGCAATGAGAGAGGAGGGAAA 112 179 Eco 179 151, 28179, 151, TCTCTGGGT TGGCG 0109 I 28 39 G12373A dGTCCCTCATTTTCCTTCAAAA113 dGGGAAGAGAATGGATTTCCT 114 174 Tsp 174 141, 33 174, 141, GCGGGCAGGGAGC R I 33

TABLE 4 Amino Acid Polymorphisms SNP POSITION LOCATION VARIANT I VARIANT2 Effect on amino acid side chain 5 A7164T EXON 1 MET LEU Conservative 6C10039T EXON 2 PRO PRO Neutral 7 C10082T EXON 2 LEU SER Hydrophobic -Hydrophilic 8 C10108T EXON 2 GLY GLY Neutral 9 G10344A EXON 2 SER ASNConservative 10 10363 (AAG)ins EXON 2 SER insertion SER deletion SERinsertion/deletion 11 A10516G EXON 2 GLN GLN Neutral 12 C10521T EXON 2SER PHE Hydrophilic - Hydrophobic 13 T10624C EXON 2 SER SER Neutral 14G10669A EXON 2 ARG ARG Neutral 15 T10873C EXON 2 TYR TYR Neutral 16G11020A EXON 2 ALA ALA Neutral 17 A11117G EXON 2 SER GLY Hydrophilic -Hydrophobic 18 T11138G EXON 2 SER ALA Hydrophilic - Hydrophobic 19G11142T EXON 2 GLY VAL Conservative 20 C11145T EXON 2 SER LEUHydrophilic - Hydrophobic 21 G11233C EXON 2 SER SER Neutral 22 T11260CEXON 2 CYS CYS Neutral 23 G11495A EXON 2 ASP ASN Hydrophilic charged -Hydrophilic neutral

TABLE 5 Primer Sequence SEQ ID Primer Sequence SEQ ID Primer NameForward NO Reverse NO SEEK INI_8 CAGTGAGCTGAGACCGTG 115CTGGTACCAGTGTGTCAG 116 SEEK INI_8 CAGTGAGCTGAGACCGTG 115CTGGTACCAGTGTGTCAG 116 SEEK INI_8 CAGTGAGCTGAGACCGTG 115CTGGTACCAGTGTGTCAG 116 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK INI_6 GACTCCTCAGAGCCTCAG 117GTAGCTACTGAAGCCGCTG 118 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM3 CCTAGATCAAGAGGCCCAG 119ACAGCAGGAGACTCGAGG 120 SEEK PROM2 CCTCAGATGCTTCATGAATGG 121GTGAAGTCAGCCGAATAGC 122 SEEK PROM2 CCTCAGATGCTTCATGAATGG 121GTGAAGTCAGCCGAATAGC 122 SEEK PROM2 CCTCAGATGCTTCATGAATGG 121GTGAAGTCAGCCGAATAGC 122 SEEK PROM2 CCTCAGATGCTTCATGAATGG 121GTGAAGTCAGCCGAATAGC 122 SEEK PROM2 CCTCAGATGCTTCATGAATGG 121GTGAAGTCAGCCGAATAGC 122 SEEK PROM2 CCTCAGATGCTTCATGAATGG 121GTGAAGTCAGCCGAATAGC 122

TABLE 6 Corneo- AC006163 desmosin Location SEQ nt nt in IUB ID SNPposition position gene Flanking Sequence Code NO 1  6,984 bp −115 5′UTRTACCACGCTCCTCCCGGCCACACCAACTTCCCCC/TGGGGCACCCACCCCCTCCACCTCTC Y 123CTCCTCTCCC 2  7,068 bp −31 5′UTRTGCCCAGGGAATGTCCAGCTCTGGCATAAAGGACCCA/GGGTGTCCTCGAGCTGCCATCAG R 124TCAGGAGGCCG 3  7,077 bp −22 5′UTRAGCTCTGGCATAAAGGACCCAGGTGTCCTC/TGAGCTGCCATCAGTCAGGAGGCCGTGCAG Y 125CCCGAGATGGGC 4  7,107 bp 9 5′UTRGAGCTGCCATCAGTCAGGAGGCCGTGCAGC/TCCGAGATGGGCTCGTCTCGGGCACCCTGG Y 126ATGGGGCGT 5  7,164 bp 66 Exon 1GCACCCTGGATGGGGCGTGTGGGTGGGCACGGGATGA/TTGGCACTGCTGCTGGCTGGTCT W 127CCTCCTGCCAGG 6  8,884 bp Intron I Intron ICTGGAGGGGCTAGGGAAGGCAGAAGGAACGCAGGT/AGAAAGAGTCATGGAGGAACCATGG W 128GGTAAGTT 7  8,906 bp Intron I Intron ICAGAAGGAACGCAGGTGAAAGAGTCATGGAGGAACCAT/CGGGGTAAGTTGGGCCTGGGGT Y 129TTTGAGCAA 8  8,931 bp Intron I Intron IGGAGGAACCATGGGGTAAGTTGGGCCTGGGGTTTTG/CAGCAAAGGAAAGGAAAGATAAGG S 130AAAGATGTGGCTC 9  9,538 bp Intron I Intron ICTGTCTCTTCAGGGTCCTTTCTTTTAGACCTAT/CTTGTTCCTGCCCCTTCTCCATTCCCT Y 131CTTCTTTT 10  9,607 bp Intron I Intron IAAAAAAATTTTAATTAAAAAACAAAATACAGAT/CGGGGTCTATGTTGCCCAGGCTGGTCT Y 132TGAACTCTGGGGCGC 11  9,608 bp Intron I Intron IAAAAAAATTTTAATTAAAAAACAAAATACAGATG/AGGGTCTATGTTGCCCAGGCTGGTCT R 133TGAACTCTGGGGCGC 12  9,647 bp Intron I Intron IGGGTCTATGTTGCCCAGGCTGGTCTTGAACTCTGGGGCG/ACATGCAATCCTCCCACCTCA R 134GCCTCCCAAAGTGCTGG 13  9,667 bp Intron I Intron ITCTTGAACTCTGGGGCGCATGCAATCCTCCCACCTCA/GGCCTCCCAAAGTGCTGGGATTA R 135CCGGCGTGAGCCACT 14  9,745 bp Intron I Intron IAGCCCCCTCTTATATTCAATGTATTCCTTTGAGGT/CCACTCACTTTGGCACGTAATTTTC Y 136TATTTTTCTGGTTG 15  9,761 bp Intron I Intron ITCAATGTATTCCTTTGAGGTCACTCACTTTGGCACG/CTAATTTTCTATTTTTCTGGTTGG S 137TGTTTGCCCACCCTT 16  9,926 bp Intron I Intron ICCCTGCGCTCTGCTTGGGAGAAACCCGAGAGGCCGATT/GACTGAGATAAGGCAGAAAGGT K 138GAGGGAGGAAGCCA 17  9,952 bp Intron I Intron IAGAGGCCGATTACTGAGATAAGGCAGAAAGGTGAGGG/AAGGAAGCCAAGCCTCTTTGGCC R 139CTTACTAACCACTG 18  9,968 bp Intron I Intron IACTGAGATAAGGCAGAAAGGTGAGGGAGGAAGCCAAGCCTCT/CTTGGCCCTTACTAACCA Y 140CTGCTTTCCTCCACAGGGACCTTG 19 10,039 bp 137 Exon 2CAGGGACCTTGGCTAAGAGCATTGGCACCTTCTCAGACCCC/TTGTAAGGACCCCACGCGT Y 141ATCACCTCCCCTAACGACCCCT 20 10,082 bp 180 Exon 2GGACCCCACGCGTATCACCTCCCCTAACGACCCCTGCC/TTCACTGGGAAGGGTGACTCCA Y 142GCGGCT 21 10,108 bp 206 Exon 2ACGACCCCTGCCTCACTGGGAAGGGTGACTCCAGCGGC/TTTCAGTAGCTACAGTGGCTCC Y 143AGCAGTTCTGGCAGCTCCAT 22 10,344 bp 442 Exon 2CCGGTTCCTCCCAGCTGGGGAGCAGCAGCTCTCACTCGGGAAG/ACAGCGGCTCTCACTCG R 144GGAAGCAGCAGCTCTCATTCG 23 10,363 bp 461 Exon 2GGAGCAGCAGCTCTCACTCGGGAAGCAGCGGCTCTCACTCGGG(AAG)CAGCAGCTCTCAT Ins/del145 (ins) TCGAGCAGCAGCAGCAGCTT 24 10,516 bp 614 Exon 2AATACTAAACCCTTCCCAGCCTGGACAAAGCTCTTCCTCTTCCCAA/GACCTYTGGGGTAT R 146CCAGCAGTGGCCAAAGCGTCAGCTCC 25 10,521 bp 619 Exon 2AATACTAAACCCTTCCCAGCCTGGACAAAGCTCTTCCTCTTCCCAAACCTC/TTGGGGTAT Y 147CCAGCAGTGGCCAAAGCGTCAGCTCC 26 103624 bp 722 Exon 2CGACTCTCCCTGCAGTGGAGGGCCCATCGTCTCGCACTCT/CGGCCCCTACATCCCCAGCT Y 148CCCACTCTGTGTC 27 10,669 bp 767 Exon 2CCTACATCCCCAGCTCCCACTCTGTGTCAGGGGGTCAGAGG/ACCTGTGGTGGTGGTGGTG R 149GACCAGCACGGTTCTGGTGC 28 10,873 bp 971 Exon 2ACAGTTATCTGGTTCCAGGCATGACCTACAGTAAGGGTAAAATCTAT/CCCTGTGGGCTAC Y 150TTCACCAAAGAGAACCCTGTGA 29 11,020 bp 1118 Exon 2ACCCCATCATCCCCAGCCAGTCGGCAGCTTCCTCGGCCATTGCG/ATTCCAGCCAGTGGGG R 151ACTGGTGGGGTCCAGC 30 11,117 bp 1215 Exon 2CCAAGGGACCCTGCTCTCCCTCCAGTTCTCGAGTCCCCAGCA/GGTTCTAGCATTTCCAGC R 152AGCTCCGGTTCACCCTA 31 11,138 bp 1236 Exon 2CTCGAGTCCCCAGCAGTTCTAGCATTTCCAGCAGCT/GCCGGTTCACCCTACCATCCCTGC K 153GGCAGTGCTT 32 11,142 bp 1240 Exon 2 CTAGCATTTCCAGCAGGTCCG G/TTTCACCCTACCATCCCTGCGGCAGTGCT K 154 33 11,145 bp 1243 Exon 2CCAGCAGTTCTAGCATTTCCAGCAGCTCCGGTTC/TACCCTACCATCCCTGCGGCAGTGCT Y 155TCCCAGAG 34 11,233 bp 1331 Exon 2GGCACCGGGTCCTTCAGCAGCAGCTCCAGTTCCCAATCG/CAGTGGCAAAATCATCCTTCA S 156GCCTTGTGGCAGCAA 35 11,260 bp 1358 Exon 2AGTTCCCAATCGAGTGGCAAAATCATCCTTCAGCCTTGT/CGGCAGCAAGTCCAGCTCTTC Y 157TGGTCACCCTTGC 36 11,495 bp 1593 Exon 2TGAAGTTTTCCTACCCCAAGGAGAGTTACTCG/AACAGTCCAT(AAG)AAGTCAACTGTTG R 158TGTGTGTGCAT 37 11,505 bp 1603 3′ UTRTACCCCAAGGAGAGTTACTCGACAGTCCAT(AAG)AAGTCAACTGTTGTGTGTGTGCATGC Ins/del159 (ins) CTTGGGCACAAA 38 11,575 bp 1674 3′ UTRGGCACAAACAAGCACATACACTATATCCCATATGGGAGAAGG/TCAGTGCCCAGGCATAGG K 160GTTAGCTCAGTTTCCCTCCTTCCCA 39 11,641 bp 1739 3′ UTRAGCTCAGTTTCCCTCCTTCCCAAAAGAGTGGTTCTGCTTTCTCT/CACTACCCTAAGGTTG Y 161CAGACTCTCTCTTATCAC 40 11,649 bp 1747 3′ UTRAAAAGAGTGGTTCTGCTTTCTCYACTACCCT/CAAGGTTGCAGACTCTCTCTTATCACCCC Y 162TTCCTCCTTCCTC 41 11,808 bp 1906 3′ UTRAGATCACCACCCCTTACAATTCCCTCTACTGTGTT/GGAAATGGTCCATTGAGTAACACCC K 163CCATCACCTTCTCAACT 42 11,839 bp 1937 3′ UTRGAAATGGTCCATTGAGTAACACCCCCATCAC/GCTTCTCAACTGGGAAACCCCTGAAATGC S 164TCTCAGAGCACC 43 11,885 bp 1963 3′ UTR TGAAATGCTCTCAGAGCACCTCTGA T/CGCCTGAAGAAGTTATACCTTCCTC Y 165 44 11,977 bp 2075 3′ UTRAACCATCATCTGGAAACAGTGGCCACTTTTCACTGACCTC/TTCTTCGACATCTAGTCAAC Y 166CCACCCAATATGC 45 12,018 bp 2116 3′ UTRATCTAGTCAACCCACCCAATATGCCACTGGGCTT/CTCGCTCCCAATTCCACCCCACCCTC Y 167CATTACAGAGCTCACCA 46 12,136 bp 2234 3′ UTRGCCTCTCAAGGCCCTTATCTCAGCCCCTTCCTGTGGCCAT/CTTCCCTCAGTGCCCAGATG Y 168ATTCCCTGGGTGAGGGCAGACAC 47 12,149 bp 2247 3′ UTRCAGCCCCTTCCTGTGGCCATTTCCCTCAGTGCC/TCAGATGATTCCCTGGGTGAGGGAGAC Y 169ACTGGGGCACCCTC 48 12,198 bp 2296 3′ UTRTTCCCTGGGTGAGGGAGACACTGGGGCACCCTCAGAGGTTGG/AAGCAGGCTCCCTGCTGT R 170CCCTGGATCCTGGACAGA 49 12,283 bp 2381 3′ UTR GGTGCAGACTTTTTGCCTTCTTGGAG/A TCCTGGGTCTCCTCTGAGAGTCTG R 171 50 12,318 bp 2416 3′ UTRTCTTGGAGTCCTGGGTCTCCTCTGAGAGTCTGGGTGGTGCTCT/CTCCTACGCCTCTAGAG Y 172GTCTCTGTGTCCCTCA 51 12,345 bp 2443 3′ UTRTGGGTGGTGCTCTTCCTACGCCTCTAGAGGTCTCTGTGTC/TCCTCATTTTCCTTCAAAAG Y 173CGGGCTGTGTTTCT 52 12,373 bp 2471 3′ UTR TCATTTTCCTTCAAAAGCGGGCTGT G/ATTTCTCTTCTACCTTCCAGCTCCT R 174 53 12,901 bp 2999 3′ UTRTAGATCAAGAGGCCCAGCCTGTGGCAGAACAGAGCTGCCA/GGTGGTCTCTCCATCTTCAC R 175ACTCCCTGCTCTGCTGGGGT 54 13,001 bp 3099 3′ UTRAACATGGCTCTCAGGTGAGGGCTGAGAAGGCAGAGTGCCCCA/CGTGGGAAAGAGGAGTCG M 176CTTCCACTGGAGAAGAGAGA 55 13,020 bp 3118 3′ UTRGCTGAGAAGGCAGAGTGCCCCAGTGGGAAAGAGGAGTCGCT/CTCCACTGGAGAAGAGAGA Y 177GAAAGTGGAGTGTGTGGTG 56 13,108 bp 3206 3′ UTRGACTTAAGTCCTGAGACAGGCAGGGAGAGGCTGAGGCGGAC/GGAAGTTCCCGCATCCCAA S 178GGAGGGCAGAGTGGATT 57 13,117 bp 3215 3′ UTRTGAGACAGGCAGGGAGAGGCTGAGGCGGACGAAGTTCCC/TGCATCCCAAGGAGGGCAGAG Y 179TGGATTGTGCTTGTCC 58 13,178 bp 3276 3′ UTRGGATTGTGCTTGTCCCTGTAGGAGCCCCACCCCCCACCCC/TAGGCCACCTCTCAGAGCCT Y 180CTGCTTGGCTGCAAAGG 59 13,224 bp 3322 3′ UTRCTCAGAGCCTCTGCTTGGCTGCAAAGGAATTCACCCC/TTACTGTAGCACTTAACCCATTC Y 181CCTCCTATCAGGGTGG 60 13,316 bp 3414 3′ UTRTGAATTTAGAACTGTTGAAACTCCAAGTCTGGAATCAGCAA/GAAATGTATTACATTGACC R 182AGAAAGGGATTGAATCACCCT 61 13,365 bp 3463 3′ UTRACATTGACCAGAAAGGGATTGAATCACCCTTGGTCCAGCA/GTCTGGCCCCTGATCTGCAG R 183CCAATGGCAGGAATCGAGGTC 62 13,562 bp 3660 3′ UTRAGGCCTCTGGGCTCCATCCACTGCCAGTTCTGGAGA/TGGAGCTCTTCACTCCTCCAGTGG W 184TTAAGCCAGCA 63 13,605 bp 3703 3′ UTRCTCTTCACTCCTCCAGTGGTTAAGCCAGCAGGGGCAGGT/CGGGGAGGACACAGCAGTAGA Y 185ATCAGCCAACAGCTCAT 64 13,670 bp 3768 3′ UTRCATGTTTAGACCTTGGGCAGCCAGGGAAGCC/TTACTCCTGGGGCCTCCCGGAAGCCATGG Y 186AGAGAAC 65 13,859 bp 3857 3′ UTRGATCAAGTCCTGGCCATTTGACAGCAGCATTTAAAGGCT/CCTCCTCTACTGTTACTTGGA Y 187AATAGCCACTTTCTCCCAAGGT 66 13,889 bp 3897 3′ UTRCTCCTCTACTGTTACTTGGAAATAGCCACT/CTTCTCCCAAGGTTTCTTATACTCT Y 188 67 13,914bp 3922 3′ UTRGAAATAGCCACTTTCTCCCAAGGTTTCTTATACTCTG/ATGGCACATCTGACCACCAGTAG R 189CAGGCAGAATGATGT

TABLE 7 AC006163 Frequency SNP nt position nt position* SNP chemistryallele 1 allele 2 1  6,984 bp 44884 CDSN6984 PSQ 69.6 30.4 2  7,068 bp44968 CDSN7068 PSQ 60.8 39.2 19 10,039 bp 47939 PS SEEK IN 1 6 C565TSequenced 55 45 21 10,108 bp 48008 CDSN C10098T Sequenced not availablenot available 22 10,344 bp 48244 CDSN G10343A Sequenced not availablenot available 23 10,363 bp (ins) 48262 CDSN 10363 AAG ins Sequenced notavailable not available 24 10,516 bp 48416 CDSNx2.2A10516G PSQ 47.8 52.225 10,521 bp 48421 CDSNx2.2C10521T PSQ 20.5 79.5 26 10,624 bp 48524CDSNx2T10614C SNaPshot 48.9 51.1 27 10,669 bp 48569 CDSNx2.2G10669ASNaPshot 85.7 14.3 28 10,873 bp 48773 CDSN T10873C SNaPshot 32.3 67.7 2911,020 bp 48920 SEEKIN1_3 G27A PSQ 43.8 56.2 30 11,117 bp 49017SEEKIN1_3 A124G PSQ 98.8 1.2 31 11,138 bp 49038 SEEKIN1_3 T145G PSQ 82.817.2 32 11,142 bp 49042 SEEKIN1_3 G149T PSQ 100 0 33 11,145 bp 49045SEEKIN1_3 C152T PSQ 64.3 35.7 34 11,233 bp 49133 SEEKIN1_3 G241C PSQ47.8 52.2 35 11,260 bp 49160 SEEKIN1_3 T268C PSQ 78.9 21.1 36 11,495 bp49395 SEEK1in3 G503A SNaPshot 68.7 31.3 37 11,605 bp (ins) 49404-49407SEEK1in3.511INS SNaPshot 43.4 56.6 38 11,575 bp 49479 CDSN G11576TSNaPshot 32.5 67.5

TABLE 8 SNP1 SNP2 SNP19 SNP21 SNP22 SNP23 SNP24 SNP25 SNP26 SNP27 SNP28SNP1 1 0.76 0.89 0.75 −0.88 −0.19 0.55 0.23 −0.56 0.13 −0.47 SNP2 1 0.810.74 −0.8 −0.3 0.45 0.18 −0.42 0.17 −0.27 −0.6 SNP19 1 0.79 −0.91 −0.260.56 0.19 −0.54 0.15 −0.41 −0.6 SNP21 1 −0.84 −0.3 0.43 0.16 −0.39 0.07−0.35 −0.48 SNP22 1 0.26 −0.56 −0.18 0.52 −0.13 0.41 0.55 SNP23 1 0.550.23 −0.55 0.17 −0.4 −0.47 SNP24 1 0.41 −0.99 0.36 −0.71 −1 SNP25 1−0.43 −0.27 −0.59 −0.33 SNP26 1 −0.33 0.71 1 SNP27 1 0.34 −0.33 SNP28 10.65 SNP29 1 SNP30 SNP31 SNP32 SNP33 SNP34 SNP35 SNP36 SNP37 SNP38 SNP29SNP30 SNP31 SNP32 SNP33 SNP34 SNP35 SNP36 SNP37 SNP37 SNP1 −0.57 −0.10.04 n/a 0.46 −0.55 0 −0.92 0.55 0.55 SNP2 0.03 0.16 n/a 0.41 −0.48 0.05−0.8 0.45 0.29 0.29 SNP19 −0.09 0.12 n/a 0.46 −0.55 0.08 −0.93 0.55 0.420.42 SNP21 −0.01 0.05 n/a 0.33 −0.39 0.11 −0.8 0.4 0.34 0.34 SNP22 0.08−0.11 n/a −0.45 0.54 −0.06 0.94 −0.53 −0.41 −0.41 SNP23 −0.14 0.14 n/a0.52 −0.5 −0.26 0.27 0.54 0.4 0.4 SNP24 −0.16 0.3 n/a 0.86 −0.96 −0.28−0.56 1 0.71 0.71 SNP25 0.02 −0.29 n/a 0.36 −0.34 −0.25 −0.2 0.43 0.580.58 SNP26 0.15 −0.28 n/a −0.86 0.94 0.22 0.55 −0.98 −0.71 −0.71 SNP270.08 0.93 n/a 0.27 −0.31 0 −0.16 0.33 −0.36 −0.36 SNP28 0.18 0.35 n/a−0.64 0.67 0.25 0.44 −0.71 −0.97 −0.97 SNP29 0.02 −0.14 n/a −0.67 1−0.34 0.61 −1 −0.72 −0.72 SNP30 1 0.1 n/a −0.15 0.13 −0.02 0.11 −0.13−0.18 −0.18 SNP31 1 n/a 0.3 −0.25 −0.01 −0.13 0.27 −0.37 −0.37 SNP32 n/an/a n/a n/a n/a n/a n/a SNP33 1 −0.82 −0.25 −0.48 0.86 0.64 0.64 SNP34 10.18 0.58 −0.95 −0.68 −0.68 SNP35 1 −0.1 −0.23 −0.23 −0.23 SNP36 1 −0.55−0.44 −0.44 SNP37 1 0.72 0.72 SNP38 1 1

TABLE 9 Number of Transmissions SNP Frequency chi- p value Allele 1Allele 2 Number Position SNP Type (allele 1) Transmissions squared(bootstrap) observed expected observed expected 1 44884 Promoter 0.79117 3.99 0.043 235 226 53 62 2 44968 Promoter 0.69 98 1.14 0.213 157 15267 72 19 47939 Silent 0.79 133 11.43 0.002 260 244 50 66 21 48008leu-ser 0.74 125 10.7 0 238 221 60 77 22 48244 ser-asn 0.2 132 3.440.061 55 64 259 250 23 48262 ins/del (ser) 0.82 112 9.28 0 231 219 33 4524 48416 Silent 0.59 125 18.03 0 203 180 99 122 25 48421 ser-phe 0.18120 1.43 0.18 44 49 232 227 26 48524 silent 0.43 140 22.93 0 113 143 229199 27 48569 silent 0.13 139 5.97 0.025 35 45 305 295 28 48773 silent0.56 142 36.51 0 152 188 194 158 29 48920 silent 0.47 26 0.99 0.283 2326 33 31 30 49017 ser-gly 0.96 131 11.16 0 291 299 21 13 31 49038ser-ala 0.13 135 4.55 0.051 34 42 290 282 32 49042 gly-val 1 33 49045ser-leu 0.59 132 9.69 0 211 193 111 129 34 49133 ser-leu 0.43 133 110.002 115 135 203 183 35 49160 silent 0.33 102 0.74 0.381 77 81 169 16536 49395 silent 0.22 140 6.47 0.02 61 74 281 268 37 49404 ins/del 0.58139 18.32 0 223 197 113 139 38 49479 3′ UTR 0.44 144 34.99 0 194 158 156192

TABLE 10a SNP Haplotype Number A B C 1 1 1 2 2 1 1 2 19 1 1 2 21 1 1 222 2 2 1 23 1 1 1 24 1 1 2 25 1 2 2 26 2 2 1 27 2 2 2 28 2 2 1 30 1 1 131 2 2 2 33 1 1 2 34 2 2 1 35 2 2 1 36 2 2 1 37 1 1 2 38 1 1 2

TABLE 10b Code Key 1 2 A/T A T A/G A G A/C A C C/G G C G/T G T C/T C T

1. A method comprising the detection of one or more variant alleles inthe corneodesmosin gene at one or more nucleotide positions selectedfrom the group consisting of 4805 and 4875 of SEQ ID NO:1.
 2. The methodof claim 1 wherein the one or more variant alleles are selected from thegroup consisting of 4805AAG (insert), and G4875T.
 3. A method fordiagnosing or determining susceptibility of a subject to acorneodesmosin-mediated disease comprising the detection of one or morevariant alleles in the corneodesmosin gene at one or more nucleotidepositions selected from the group consisting of 3339, 3408, 4805, and4875 of SEQ ID NO:1.
 4. The method of claim 3 wherein the one or morevariant alleles are selected from C3339T, C3408T, 4805AAG (insert), andG4875T.
 5. The method of claim 1 or 3 wherein thecorneodesmosin-mediated disease is an inflammatory disease
 6. The methodof claim 1 or 3 wherein the corneodesmosin-mediated disease ispsoriasis.
 7. The method of claim 1 or 3 wherein detection of the one ormore variant alleles is carried out using a polynucleotide isolated froma biological sample selected from the group consisting of whole blood,semen, saliva, tears, skin, hair, and a buccal sample.
 8. The method ofclaim 1 or 3 wherein detection of the one or more variant alleles iscarried out using an agent capable of detecting one or more of thevariant alleles.
 9. The method of claim 8 wherein the agent is ananti-sense polynucleotide that is complementary to one or more of thevariant alleles or to a region flanking one or more of the variantalleles.
 10. The method of claim 1 or 3 wherein detection of one or moreof the variant alleles is carried out using a method selected from thegroup consisting of direct probing, allele specific hybridization,polymerase chain reaction (PCR), allele specific amplification (ASA),and restriction fragment length polymorphism (RFLP).
 11. The method ofclaim 1 or 3 wherein detection of one or more of the variant alleles iscarried out by detecting the presence of a variant protein.
 12. Themethod of claim 11 wherein detection of the variant protein is carriedout using an antibody to an antigen of the variant protein.
 13. An agentcapable of detecting one or more variant alleles in the corneodesmosingene at one or more nucleotide positions selected from the groupconsisting of 3339, 3408, 4805, and 4875 of SEQ ID NO:1.
 14. The agentof claim 13 wherein the one or more variant alleles are selected fromthe group consisting of C3339T, C3408T, 4805AAG (insert), and G4875T.15. The agent of claim 13 for use in a method for the diagnosis of ordetermining the susceptibility of a subject to a corneodesmosin-mediateddisease.
 16. The agent of claim 13 wherein the agent is: (a) ananti-sense polynucleotide that is complementary to one or more of thevariant alleles or to a region flanking one or more of the variantalleles; or (b) an antibody to an antigen of the protein expressed fromone or more of the variant corneodesmosin genes.
 17. A method ofscreening for an agent capable of detecting one or more variant allelesin the corneodesmosin gene at one or more nucleotide positions selectedfrom the group consisting of 3339, 3408, 4805, and 4875 of SEQ ID NO:1,wherein the method is selected from the group consisting of: (i) amethod comprising the steps of contacting a putative agent with apolynucleotide comprising one or more variant alleles in thecorneodesmosin gene at one or more nucleotide positions selected fromthe group consisting of 3339, 3408, 4805, and 4875 of SEQ ID NO:1, andmonitoring the reaction there between; and (ii) a method comprising thesteps of contacting a putative agent with a protein expressed from oneor more variant alleles of the corneodesmosin gene having an alterationat one or more nucleotide positions selected from the group consistingof 3339, 3408, 4805, and 4875 of SEQ ID NO:1, and monitoring thereaction there between.
 18. The method of claim 17 wherein the one ormore variant alleles are selected from C3339T, C3408T, 4805AAG (insert),and G4875T.
 19. The method of claim 17 further comprising: (a)contacting the putative agent with a polynucleotide having SEQ ID NO:1,and comparing the reaction between: (i) the putative agent and thepolynucleotide comprising the variant allele; and (ii) the putativeagent and the polynucleotide having SEQ ID NO:1; or (b) contacting theputative agent with a protein having SEQ ID NO:2 and comparing thereaction between: (i) the putative agent and the protein expressed fromthe polynucleotide comprising the variant allele; and (ii) the putativeagent and the protein having SEQ ID NO:2.
 20. The method of claim 17wherein the method is carried out by contacting the putative agent witha host cell or transgenic non-human animal comprising the polynucleotidecomprising the variant allele or the polypeptide expressed from thepolynucleotide comprising the variant allele.
 21. A kit for thedetection of a variant allele in the corneodesmosin gene comprising anagent capable of detecting one or more variant alleles in thecorneodesmosin gene at one or more nucleotide positions selected fromthe group consisting of 3339, 3408, 4805, and 4875 of SEQ ID NO:1. 22.The kit of claim 21 wherein the one or more variant alleles are selectedfrom C3339T, C3408T, 4805AAG (insert), and G4875T.
 23. The kit of claim21 wherein the agent is selected from the group consisting of: (a) ananti-sense polynucleotide that is complementary to one or more of thevariant alleles or to a region flanking one or more of the variantalleles; (b) an antibody to an antigen of the protein expressed from thecorneodesmosin gene comprising one or more of the variant alleles; and(c) a restriction enzyme that is capable of detecting the presence ofthe polynucleotide comprising one or more of the variant alleles. 24.The kit of claim 21 further comprising means for the detection of areaction selected from the group consisting of: (a) nucleotide detectionmeans; (b) labeled secondary antibodies; and (c) size detection means.25. The kit of claim 21 for use in a method for the diagnosis of ordetermining the susceptibility of a subject to a corneodesmosin-mediateddisease, and further comprising a key correlating the presence of theone or more variant alleles with the presence of, or susceptibility to,corneodesmosin-mediated disease.